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Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bonocora RP, Smith C, Lapierre P, Wade JT - PLoS Genet. (2015)

Bottom Line: Strikingly, the majority of σ54 binding sites are located inside genes.We conclude that many intragenic σ54 binding sites are likely to be functional.Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

ABSTRACT
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

No MeSH data available.


Related in: MedlinePlus

Validation of intragenic σ54 promoters for the nagE and yqeB mRNAs.(A) Intragenic PnagB and PyqeC σ54 promoters were chromosomally mutated. The sequence of both σ54 promoters is shown; conserved, consensus residues (blue text) and the mutagenic changes (red text) are indicated. (B) The relative σ54 occupancy compared to a positive control region (the σ54 promoter of glnA) was measured by ChIP-qPCR at both promoters in wild-type (RPB220 for nagB and RPB232 for yqeB; black bars), ΔPnagB (RPB277; white bars) and ΔPyqeC (RPB279; white bars) E. coli strains. Note that “wild-type” and “mutant” refer to the status of the promoter. Strains used to evaluate PnagB and NagE contain a C-terminal FLAG-tagged nagE gene, whereas the strains used to evaluate PyqeC and YqeB contain a C-terminal FLAG-tagged yqeB gene. (C) Expression of nagE and yqeB, the genes immediately downstream of PnagB and PyqeC σ54 promoters, respectively, relative to the expression of glnA, was measured by RT-qPCR. (D) Western blot probing of extracts from NagE and YqeB FLAG-tagged and untagged E. coli strains with anti-FLAG antibody. Probing the same membranes with anti-α (RNAP subunit) antibody served as a loading control. Wild-type (+) and mutated (-) promoters are indicated. The blot is representative of three independent biological replicates.
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pgen.1005552.g007: Validation of intragenic σ54 promoters for the nagE and yqeB mRNAs.(A) Intragenic PnagB and PyqeC σ54 promoters were chromosomally mutated. The sequence of both σ54 promoters is shown; conserved, consensus residues (blue text) and the mutagenic changes (red text) are indicated. (B) The relative σ54 occupancy compared to a positive control region (the σ54 promoter of glnA) was measured by ChIP-qPCR at both promoters in wild-type (RPB220 for nagB and RPB232 for yqeB; black bars), ΔPnagB (RPB277; white bars) and ΔPyqeC (RPB279; white bars) E. coli strains. Note that “wild-type” and “mutant” refer to the status of the promoter. Strains used to evaluate PnagB and NagE contain a C-terminal FLAG-tagged nagE gene, whereas the strains used to evaluate PyqeC and YqeB contain a C-terminal FLAG-tagged yqeB gene. (C) Expression of nagE and yqeB, the genes immediately downstream of PnagB and PyqeC σ54 promoters, respectively, relative to the expression of glnA, was measured by RT-qPCR. (D) Western blot probing of extracts from NagE and YqeB FLAG-tagged and untagged E. coli strains with anti-FLAG antibody. Probing the same membranes with anti-α (RNAP subunit) antibody served as a loading control. Wild-type (+) and mutated (-) promoters are indicated. The blot is representative of three independent biological replicates.

Mentions: To further investigate the intragenic σ54 binding sites within nagB and yqeC, we constructed strains with epitope tags fused to 3ʹ ends of nagE or yqeB. We then constructed derivatives of these strains with mutations in the σ54 binding site inside nagB, or yqeC, respectively (one silent change and one His → Lys codon change in nagB; two silent changes in yqeC; Fig 7A). We measured association of σ54 with the wild type and mutated sites using ChIP-qPCR. Our data indicate that mutating the putative binding sites greatly reduces binding of σ54, confirming that these are genuine σ54 binding sites (Fig 7B). The microarray data described above strongly suggested that each of these intragenic σ54 binding sites is a promoter for an mRNA for the downstream gene. To test this hypothesis, we used qRT-PCR to measure mRNA levels of the downstream gene for each putative promoter (nagE and yqeB) in wild type cells and cells in which the binding site is disrupted (Fig 7C). These data indicate that mutation of either σ54 binding site results in a large decrease in the mRNA level for the downstream gene. Lastly, we used Western blotting with an antibody specific to the epitope tags to measure NagE and YqeB protein levels in cells with wild type and mutant promoters. Consistent with the qRT-PCR data, mutation of either promoter resulted in a decrease in the protein level for the downstream gene (Fig 7D). In the case of NagE, the decrease in protein level was modest (~2-fold), whereas YqeB was undetectable in the promoter mutant. We conclude that the σ54 binding sites within nagB and yqeC represent promoters for nagE and yqeB, respectively, with the mRNAs having unusually long 5ʹ UTRs.


Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bonocora RP, Smith C, Lapierre P, Wade JT - PLoS Genet. (2015)

Validation of intragenic σ54 promoters for the nagE and yqeB mRNAs.(A) Intragenic PnagB and PyqeC σ54 promoters were chromosomally mutated. The sequence of both σ54 promoters is shown; conserved, consensus residues (blue text) and the mutagenic changes (red text) are indicated. (B) The relative σ54 occupancy compared to a positive control region (the σ54 promoter of glnA) was measured by ChIP-qPCR at both promoters in wild-type (RPB220 for nagB and RPB232 for yqeB; black bars), ΔPnagB (RPB277; white bars) and ΔPyqeC (RPB279; white bars) E. coli strains. Note that “wild-type” and “mutant” refer to the status of the promoter. Strains used to evaluate PnagB and NagE contain a C-terminal FLAG-tagged nagE gene, whereas the strains used to evaluate PyqeC and YqeB contain a C-terminal FLAG-tagged yqeB gene. (C) Expression of nagE and yqeB, the genes immediately downstream of PnagB and PyqeC σ54 promoters, respectively, relative to the expression of glnA, was measured by RT-qPCR. (D) Western blot probing of extracts from NagE and YqeB FLAG-tagged and untagged E. coli strains with anti-FLAG antibody. Probing the same membranes with anti-α (RNAP subunit) antibody served as a loading control. Wild-type (+) and mutated (-) promoters are indicated. The blot is representative of three independent biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591121&req=5

pgen.1005552.g007: Validation of intragenic σ54 promoters for the nagE and yqeB mRNAs.(A) Intragenic PnagB and PyqeC σ54 promoters were chromosomally mutated. The sequence of both σ54 promoters is shown; conserved, consensus residues (blue text) and the mutagenic changes (red text) are indicated. (B) The relative σ54 occupancy compared to a positive control region (the σ54 promoter of glnA) was measured by ChIP-qPCR at both promoters in wild-type (RPB220 for nagB and RPB232 for yqeB; black bars), ΔPnagB (RPB277; white bars) and ΔPyqeC (RPB279; white bars) E. coli strains. Note that “wild-type” and “mutant” refer to the status of the promoter. Strains used to evaluate PnagB and NagE contain a C-terminal FLAG-tagged nagE gene, whereas the strains used to evaluate PyqeC and YqeB contain a C-terminal FLAG-tagged yqeB gene. (C) Expression of nagE and yqeB, the genes immediately downstream of PnagB and PyqeC σ54 promoters, respectively, relative to the expression of glnA, was measured by RT-qPCR. (D) Western blot probing of extracts from NagE and YqeB FLAG-tagged and untagged E. coli strains with anti-FLAG antibody. Probing the same membranes with anti-α (RNAP subunit) antibody served as a loading control. Wild-type (+) and mutated (-) promoters are indicated. The blot is representative of three independent biological replicates.
Mentions: To further investigate the intragenic σ54 binding sites within nagB and yqeC, we constructed strains with epitope tags fused to 3ʹ ends of nagE or yqeB. We then constructed derivatives of these strains with mutations in the σ54 binding site inside nagB, or yqeC, respectively (one silent change and one His → Lys codon change in nagB; two silent changes in yqeC; Fig 7A). We measured association of σ54 with the wild type and mutated sites using ChIP-qPCR. Our data indicate that mutating the putative binding sites greatly reduces binding of σ54, confirming that these are genuine σ54 binding sites (Fig 7B). The microarray data described above strongly suggested that each of these intragenic σ54 binding sites is a promoter for an mRNA for the downstream gene. To test this hypothesis, we used qRT-PCR to measure mRNA levels of the downstream gene for each putative promoter (nagE and yqeB) in wild type cells and cells in which the binding site is disrupted (Fig 7C). These data indicate that mutation of either σ54 binding site results in a large decrease in the mRNA level for the downstream gene. Lastly, we used Western blotting with an antibody specific to the epitope tags to measure NagE and YqeB protein levels in cells with wild type and mutant promoters. Consistent with the qRT-PCR data, mutation of either promoter resulted in a decrease in the protein level for the downstream gene (Fig 7D). In the case of NagE, the decrease in protein level was modest (~2-fold), whereas YqeB was undetectable in the promoter mutant. We conclude that the σ54 binding sites within nagB and yqeC represent promoters for nagE and yqeB, respectively, with the mRNAs having unusually long 5ʹ UTRs.

Bottom Line: Strikingly, the majority of σ54 binding sites are located inside genes.We conclude that many intragenic σ54 binding sites are likely to be functional.Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

ABSTRACT
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

No MeSH data available.


Related in: MedlinePlus